by Lee Sau Yat. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 89-94). / Acknowledgements --- p.I / Abbreviation --- p.II / Abstract --- p.III / Table of content --- p.IV / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Historical background in the studies of LDL and LDLR --- p.1 / Chapter 1.2 --- Homeostasis of Cholesterol in Man --- p.4 / Chapter 1.2.1 --- Origin and catabolism of low density lipoprotein --- p.4 / Chapter 1.2.2 --- The LDL receptor --- p.6 / Chapter 1.2.3 --- LDL pathway --- p.7 / Chapter 1.2.4 --- Feedback regulatory action of LDL receptor --- p.10 / Chapter 1.3 --- Gene structure of LDL receptor promoter --- p.11 / Chapter 1.3.1 --- The LDL receptor promoter --- p.11 / Chapter 1.3.2 --- The responsive element in LDL receptor promoter --- p.13 / Chapter 1.4 --- Eukaryotic transcription factor --- p.15 / Chapter 1.5 --- Role of Gel-shifted assay in studying DNA binding protein --- p.17 / Chapter 1.6 --- Objective of the present thesis --- p.20 / Chapter Chapter 2 --- Materials and Methods --- p.21 / Chapter 2.1 --- Oligonucleotide synthesis and purification --- p.21 / Chapter 2.1.1 --- Primer construction --- p.21 / Chapter 2.1.2 --- Purification of oligonucleotides --- p.22 / Chapter 2.2 --- Recombinant plasmid construction --- p.24 / Chapter 2.2.1 --- Preparation of competent cell --- p.24 / Chapter 2.2.2 --- Preparation of phage DNA --- p.24 / Chapter 2.2.3 --- Amplification and purification of LDLR-promoter by PCR techniques --- p.26 / Chapter 2.2.3.1 --- Amplification and restriction site construction of LDLR- promoter --- p.26 / Chapter 2.2.3.2 --- Purification of the PCR product --- p.27 / Chapter 2.2.4 --- Preparation of plasmid pGCAT-A --- p.27 / Chapter 2.2.5 --- Recombinant plasmid pLDLRP-GCAT- A construction --- p.29 / Chapter 2.2.6 --- Transformation of DNA to competent cell --- p.29 / Chapter 2.2.7 --- Screening of positive clone pLDLRP- GCAT-A --- p.30 / Chapter 2.3 --- DNA sequencing --- p.31 / Chapter 2.3.1 --- Denaturing the double strand template --- p.31 / Chapter 2.3.2 --- Annealing reaction --- p.31 / Chapter 2.3.3 --- Labeling reaction --- p.32 / Chapter 2.3.4 --- Termination reaction --- p.32 / Chapter 2.3.5 --- Running and fixing the gel --- p.32 / Chapter 2.4 --- Cell culture and passage of different cell lines --- p.34 / Chapter 2.4.1 --- "Routine subculture of HepG 2, CHO, FSF, Cos-7 and FSF" --- p.34 / Chapter 2.4.2 --- "BMN, RTGH-1" --- p.34 / Chapter 2.5 --- Preparation of human LDL and LPDS --- p.36 / Chapter 2.5.1 --- Purification of LDL --- p.36 / Chapter 2.5.2 --- Purification of LPDS --- p.37 / Chapter 2.6 --- DNA transfection and CAT assay --- p.38 / Chapter 2.6.1 --- Transfection of recombinant plasmid to eukaryotic cells --- p.38 / Chapter 2.6.2 --- CAT assay --- p.39 / Chapter 2.7 --- 125I-LDL binding assay --- p.41 / Chapter 2.7.1 --- Radioactive iodination of LDL --- p.41 / Chapter 2.7.2 --- Purification of iodinated LDL --- p.41 / Chapter 2.7.3 --- Down regulation of LDL receptorin different cell lines --- p.41 / Chapter 2.7.4 --- Different Drugs treatment in HepG2 Cell line --- p.42 / Chapter 2.7.5 --- 125I-LDL binding assay --- p.43 / Chapter 2.8 --- Gel-shift mobility assay --- p.44 / Chapter 2.8.1 --- Extraction of crude nuclear extracts --- p.44 / Chapter 2.8.2 --- 5'end-labeling of synthetic oligonucleotides --- p.45 / Chapter 2.8.3 --- Purification of labeled oligonucleotides --- p.45 / Chapter 2.8.4 --- Nuclear protein and DNA binding reaction --- p.46 / Chapter 2.8.5 --- Gel-shift mobility electrophoresis by PhastSystem --- p.46 / Chapter 2.9 --- Construction of λ gt 11 cDNA library of HepG 2cell --- p.48 / Chapter 2.9.1 --- Purification of mRNA from HepG 2 --- p.48 / Chapter 2.9.2 --- cDNA preparation --- p.48 / Chapter 2.9.3 --- In-vitro packaging of phage --- p.48 / Chapter 2.9.3 --- Screening the expression library --- p.49 / Chapter Chapter 3 --- Results --- p.50 / Chapter 3.1 --- Construction of recombinant plasmid --- p.50 / Chapter 3.1.1 --- hLDLR-promoter λ 34 clone --- p.50 / Chapter 3.1.2 --- Restriction site generation in LDLR- promoter by PCR --- p.52 / Chapter 3.1.3 --- Preparation of pGCAT-A reporter plasmid --- p.55 / Chapter 3.1.4 --- Screening of pGCAT-A-LDLRP recombinant --- p.55 / Chapter 3.1.5 --- Sequencing of pGCAT-A-LDLR- promoter recombinant --- p.57 / Chapter 3.2 --- CAT assay of recombinant plasmid on transfected HepG 2 cell --- p.57 / Chapter 3.3 --- 125I-LDL binding assay --- p.57 / Chapter 3.3.1 --- 125 I - LDL binding assay of different cell lines --- p.57 / Chapter 3.3.2 --- Characterization of cell surface receptor of HepG 2 cell by different drugs treatment --- p.61 / Chapter 3.4 --- Gel shift mobility assay --- p.63 / Chapter 3.4.1 --- Binding effect of Repeat 2 to different cell lines --- p.63 / Chapter 3.4.2 --- Optimizing the binding reactionin HepG 2 cell by poly(dI.dC) --- p.63 / Chapter 3.4.3 --- Specificity of Repeat 2 in binding to HepG 2 cell nuclear protein --- p.66 / Chapter 3.4.4 --- LDL dose response treatment in HepG2 cell --- p.68 / Chapter 3.4.4.1 --- Binding of Repeat 2 to specific nuclear protein --- p.68 / Chapter 3.4.4.2 --- Binding of Repeat 2 to a non- specific cell nuclear protein from cells treated with LDL --- p.68 / Chapter 3.4.5 --- Effect of different drugs on the binding of Repeat 2 to nuclear proteins in HepG 2 cell --- p.71 / Chapter 3.5 --- HepG 2 cell cDNA library screening --- p.73 / Chapter Chapter 4 --- Discussion --- p.77 / Chapter 4.1 --- Strategy on construction of reporter plasmid pLDLRP-GC AT-A --- p.77 / Chapter 4.2 --- The expression of CAT in HepG 2 cell --- p.78 / Chapter 4.3 --- Identification of a DNA binding protein for Repeat 2 in HepG 2 cell --- p.80 / Chapter 4.3.1 --- Binding effect of nuclear protein to Repeat 2 --- p.82 / Chapter 4.3.1.1 --- LDL dose response relationships --- p.82 / Chapter 4.3.1.2 --- Effect of protein inhibitors --- p.83 / Chapter 4.3.1.3 --- Effect of Shanzha --- p.86 / Chapter 4.3.2 --- Screening of the cDNA library of HepG 2 cells --- p.86 / Chapter Chapter 5 --- Further Studies --- p.88 / References --- p.89 / Appendix --- p.95

Este trabalho apresenta as propriedades ópticas dos complexos Európio Tetraciclinas (EuTcs) na presença de LDL e de LDL oxidada com potenciais aplicações em análises clínicas. Foram escolhidos quatro elementos da família das Tetraciclinas: Tetraciclina (Tc), Clorotetraciclina (CTc), Metatetraciclina (MTc) e Oxitetraciclina (OTc) para fazerem parte dos complexos com o íon európio. As melhores condições para se formar os complexos eficientemente foram determinadas, através das medidas dos parâmetros ópticos como: absorção, emissão e de tempo de vida. As melhores concentrações de európio nos complexos EuTcs e possíveis influências de íons inorgânicos normalmente presentes no plasma sanguíneo também foram analisadas. As amostras foram preparadas em pH neutro e a luminescência visível do lantanídeo foi detectada após tempo de repouso das amostras de 15 minutos. Os resultados deste trabalho mostraram que as moléculas de LDL e de LDL oxidada apresentaram um importante papel no aumento da intensidade de emissão dos complexos das Tcs. As medidas realizadas com os complexos EuTcs não apresentaram deslocamentos nos comprimentos de onda dos espectros de absorção e de emissão na presença de LDL, o que demonstra a ausência de interação direta entre as moléculas de Tcs e as moléculas de LDL e LDL oxidada. No entanto, o íon európio pode interagir em diferentes sítios das moléculas de tetraciclinas o que diferenciou a intensidade de emissão de cada complexo. Comparando os resultados obtidos entre os complexos de EuTcs, o complexo EuTc foi o que apresentou perspectivas promissoras na quantificação de LDL e LDL oxidada. / This work presents the optical properties of europium complexes - Tetracyclines (EuTcs) in the presence of LDL and oxidized LDL with potential applications in clinical analysis. Four elements were chosen from the Tetracyclines family: Tetracycline (Tc), Chlortetracycline (CTc), Metatetraciclina (MTc) and Oxytetracycline (OTc) to be part of complexes with europium ion. The best conditions to form the complex efficiently were determined through measurements of optical parameters such as absorption, emission and lifetime. The best concentrations of europium complexes in EuTcs and possible influences of inorganic ions normally present in blood plasma were also analyzed. The samples were prepared at neutral pH and the visible luminescence of lanthanide was detected after resting time of the samples of 15 minutes. These results showed that the molecules of LDL and oxidized LDL have an important role in increase of the emission intensity for Tcs complexes . The measurements performed with the complex EuTcs showed no shifts in the wavelengths of the absorption and emission spectra in the presence of LDL, which demonstrates the absence of direct interaction between the molecules of Tcs and the molecules of LDL and oxidized LDL. However, the europium ion can interact at different sites of the tetracyclines molecules which differed the emission intensity of each complex. Comparing the results obtained between the complexes EuTcs, the complex EuTc is the one that presented the promising prospects in the quantification of LDL and oxidized LDL.

Este trabalho apresenta as propriedades ópticas dos complexos Európio Tetraciclinas (EuTcs) na presença de LDL e de LDL oxidada com potenciais aplicações em análises clínicas. Foram escolhidos quatro elementos da família das Tetraciclinas: Tetraciclina (Tc), Clorotetraciclina (CTc), Metatetraciclina (MTc) e Oxitetraciclina (OTc) para fazerem parte dos complexos com o íon európio. As melhores condições para se formar os complexos eficientemente foram determinadas, através das medidas dos parâmetros ópticos como: absorção, emissão e de tempo de vida. As melhores concentrações de európio nos complexos EuTcs e possíveis influências de íons inorgânicos normalmente presentes no plasma sanguíneo também foram analisadas. As amostras foram preparadas em pH neutro e a luminescência visível do lantanídeo foi detectada após tempo de repouso das amostras de 15 minutos. Os resultados deste trabalho mostraram que as moléculas de LDL e de LDL oxidada apresentaram um importante papel no aumento da intensidade de emissão dos complexos das Tcs. As medidas realizadas com os complexos EuTcs não apresentaram deslocamentos nos comprimentos de onda dos espectros de absorção e de emissão na presença de LDL, o que demonstra a ausência de interação direta entre as moléculas de Tcs e as moléculas de LDL e LDL oxidada. No entanto, o íon európio pode interagir em diferentes sítios das moléculas de tetraciclinas o que diferenciou a intensidade de emissão de cada complexo. Comparando os resultados obtidos entre os complexos de EuTcs, o complexo EuTc foi o que apresentou perspectivas promissoras na quantificação de LDL e LDL oxidada. / This work presents the optical properties of europium complexes - Tetracyclines (EuTcs) in the presence of LDL and oxidized LDL with potential applications in clinical analysis. Four elements were chosen from the Tetracyclines family: Tetracycline (Tc), Chlortetracycline (CTc), Metatetraciclina (MTc) and Oxytetracycline (OTc) to be part of complexes with europium ion. The best conditions to form the complex efficiently were determined through measurements of optical parameters such as absorption, emission and lifetime. The best concentrations of europium complexes in EuTcs and possible influences of inorganic ions normally present in blood plasma were also analyzed. The samples were prepared at neutral pH and the visible luminescence of lanthanide was detected after resting time of the samples of 15 minutes. These results showed that the molecules of LDL and oxidized LDL have an important role in increase of the emission intensity for Tcs complexes . The measurements performed with the complex EuTcs showed no shifts in the wavelengths of the absorption and emission spectra in the presence of LDL, which demonstrates the absence of direct interaction between the molecules of Tcs and the molecules of LDL and oxidized LDL. However, the europium ion can interact at different sites of the tetracyclines molecules which differed the emission intensity of each complex. Comparing the results obtained between the complexes EuTcs, the complex EuTc is the one that presented the promising prospects in the quantification of LDL and oxidized LDL.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em Farmácia / Made available in DSpace on 2012-10-26T05:24:48Z (GMT). No. of bitstreams: 1 289967.pdf: 1658757 bytes, checksum: 5a241699e8a8e7cfa5d6aaf3d0453fb8 (MD5)

Lipoprotein binding and metabolism in actively-dividing (subconfluent) and quiescent (postconfluent) bovine aortic endothelial cells (ECs) were qualitatively investigated by fluorescence microscopy using dioctadecylindocarbocyanine-labelled lipoproteins and by indirect immunofluorescence microscopy. LDL and acetylated-LDL (AcLDL) were seen bound to the surfaces of subconfluent ECs (at 4°C or at 37°C), as a random distribution of punctate foci. ECs therefore closely resembled fibroblasts in the distribution of LDL receptors on their surfaces. No binding of LDL was seen on postconfluent EC surfaces by either direct or indirect fluorescence microscopy. The patterns of AcLDL binding on postconfluent ECs resembled those on subconfluent ECs. Intracellular LDL and AcLDL occurred as perinuclear accumulations of large fluorescent disc-shaped profiles in subconfluent ECs. These accumulations were shown to arise from surface-bound material by pulse-chase experiments. Intracellular LDL was absent in the majority of postconfluent ECs, while AcLDL accumulation was massive. "Wounding" of cultures allowed simultaneous assessment of lipoprotein metabolism in quiescent and actively-dividing areas of the same culture. Quantitative assessments of the above-mentioned phenomena were made using ¹²⁵I-labelled lipoproteins. Receptor-mediated binding of LDL decreased five to ten-fold as the cultures modulated from subconfluent to postconfluent morphology. No receptor-bound LDL was detected in postconfluent ECs. Conversely, the amount of AcLDL bound increased at least fivefold during EC growth in parallel cultures. The amounts of lipoproteins endocytosed and metabolised were generally related proportionately to the amounts bound in each case. The distribution of LDL receptors on cultured cells was also investigated at the ultrastructural level using colloidal gold-conjugated LDL as a probe, and similarly labelled antibodies as probes. Whole-mounted cells with receptor probes bound to them were examined directly in the transmission electron microscope. The topographical distribution of LDL receptors has not been investigated by these techniques before. A novel method of preparing cytochemically-labelled, whole-mounted cells from styrene culture dishes was developed and used in this study. LDL Receptors expressed on the surfaces of human skin fibroblasts served to standardise these colloidal gold techniques and fortuitously led to new information on receptor distribution. Normal (FGo) and LDL receptor-negative mutant fibroblasts (GM 2000) acted as positive and negative controls respectively. Normal fibroblast LDL receptors were grouped into clusters consistent in size with coated pits (200 - 500 nm in diameter). A novel finding was the presence of a diffuse population of receptors scattered randomly amongst the clustered receptors. Another mutant fibroblast, GM 2408A, known to have an aberrant LDL receptor distribution, was also examined. Its receptors were shown to be dispersed singly, and in occasional groups of two and three, at random over the cell surfaces. No clusters were detected. The receptor-negative GM 2000 bound virtually no probes. While not as sensitive as the colloidal gold-conjugated LDL probe, an antireceptor monoclonal antibody (IgG-C7), localised by indirect immunogold labelling, gave similar results when applied to the above cells. This was taken as strong corroborative evidence that the LDL receptor distributions as determined by colloidal gold-conjugated LDL were correct. It is suggested that the dispersed population of receptors on normal fibroblasts may represent newly-emerged recycling receptors which have yet to cluster in coated pits. A further new finding reported here is the existence of the same two patterns of LD L receptors, dispersed and clustered, on the surface of subconfluent ECs. It was noted, from the study of whole-mounted and thin-sectioned cells, that the receptors were preferentially arranged in rings following the circumference of coated pit areas on the cell surface. Often these rings associated in groups or even coalesced into compound clusters. The significance of these groupings is not yet understood. In sharp contrast to the situation on subconfluent ECs, no LDL receptors (probed with the extremely sensitive colloidal-gold conjugated LDL) could be detected at the EM level on the surface of postconfluent ECs. Active cells in wounded postconfluent monolayers expressed abundant receptors detected at the EM level. It is concluded that postconfluent quiescent bovine aortic ECs in vitro metabolise virtually no LDL via the LDL-receptor pathway due to a vanishingly low number of LDL receptors. This contrasts with the ability of postconfluent cells to metabolise relatively large amounts of AcLDL via a receptor-mediated mechanism. The significance of these conclusions is discussed with respect to the interaction of plasma lipoproteins with the endothelium in vivo.

A obesidade é considerada uma doença crônica e multifatorial, onde eventos como a inflamação de baixa intensidade e as modificações oxidativas estão presentes. A elevada prevalência de obesidade tem impacto direto no desenvolvimento precoce de diabetes mellitus, hipertensão e outros fatores de risco cardiovasculares. Esse perfil tem motivado a identificação de biomarcadores precoces, sendo o monitoramento da lipoproteína de baixa densidade oxidada (LDLox) e lipoproteína de baixa densidade eletronegativa [LDL(-)] potenciais candidatos. Diante do exposto, o objetivo deste estudo foi realizar a análise comparativa e de correlação entre o conteúdo de LDLox e [LDL(-)] em adolescentes. Foram selecionados 137 adolescentes de ambos sexos, com faixa etária de 10 a 19 anos e regularmente em matriculados em escolas públicas da cidade de São Paulo. O peso, altura e circunferência da cintura (CC) foram avaliados. Após jejum (12h-15h) foi coletada uma amostra de sangue e, a partir do plasma, foram realizadas as seguintes análises: glicose, insulina, perfil lipídico, apolipoproteína (AI e B), ácidos graxos não esterificados, tamanho de HDL, atividade da CETP e LDL(-) e LDLox. Os resultados encontrados foram analisados por meio do programa SPSS 15.0, considerando valor de significância de p< 0,05. Os 137 adolescentes foram distribuídos em dois grupos: 71 no Eutrófico (51,82%) e 66 no Obeso (48,18%), segundo a classificação do IMC. 48 (35,04%) dos adolescentes eram do sexo masculino e 89 (64,96%) do sexo feminino, com idade média de 14,2 (2,3) anos. Em relação à CC, observou-se que essa confirmou a classificação feita pelo IMC. Observou-se também uma maior prevalência de hipertensão (65% p = 0,011) e obesidade (64,7% p=0,041) nos antecedentes familiares do grupo Obeso quando comparado ao grupo Eutrófico. Os adolescentes obesos apresentaram maiores valores de triglicerídeos, HDL, APO B, CETP, insulina e LDL(-) e LDLox, quando comparados aos eutróficos. Perfil inverso foi observado para Apo AI. O conteúdo de LDLox e LD(-) variou significativamente em função do IMC. Entretanto, essas partículas de LDLs não se correlacionaram entre si, embora tenham apresentado associação com outros parâmetros cardiometabólicos. Os resultados obtidos confirmam o impacto negativo da obesidade sobre os parâmetros cardiometabólicos de adolescentes e, apesar do conteúdo de LDLox e LDL(-) ter aumentado em função do IMC, essas partículas parecem ser estruturalmente distintas. Essa possibilidade foi reforçada pelas diferentes associações dessas partículas com outros marcadores bioquímicos. / Obesity is considered a chronic and multifactorial disease, where events such as low intensity inflammation and oxidative modifications are present. The high prevalence of obesity has a direct impact on the early development of diabetes mellitus, hypertension and other cardiovascular risk factors. This profile has motivated the identification of early biomarkers, and monitoring the oxidized low density lipoprotein (oxLDL) and electronegative low-density lipoprotein [LDL (-)] are potential markers. The aim of this study was to conduct a comparative analysis and correlation between the content of oxLDL and [LDL (-)] in adolescents. We selected 137 adolescents of both sexes, aged 10-19 years, enrolled in public schools in the city of São Paulo. The weight, height, waist circumference (WC) were assessed. After fasting (12-15h) samples of blood were collected and from plasma were performed the following analyzes: glucose, insulin, lipid profile, apolipoprotein (AI and B), NEFA, HDL size, CETP activity and LDL (-) and oxLDL. The results were analyzed by using SPSS 15.0 considering significant value of p <0.05. The 137 adolescents were divided into two groups: 71 Normal Weight (51.82%) and 66 Obese (48.18%), according to BMI classification. 48 (35.04%) of the adolescents were male and 89 (64.96%) females with a mean age of 14.2 (2.3) years. Regarding the CC observed, it confirmed the classification made by BMI. There was a higher prevalence of hypertension (65% p = 0.011) and obesity (64.7% p = 0.041) in the family history group Obese when compared to normal weight. Obese adolescents had higher triglyceride, HDL, APO B, CETP, insulin and LDL (-) and oxLDL, compared to normal weight. Reverse profile was observed for Apo AI. The content of oxLDL and LDL (-) varied significantly according to BMI. However, these LDL particles were not correlated with each other, although they showed cardiometabolic combination with other parameters. The results confirm the negative impact of obesity on cardiometabolic parameters of teenagers and although the content of oxLDL and LDL (-) increased as a function of BMI, these particles appear to be structurally distinct. This possibility was reinforced by different associations of these particles with other biochemical markers.

A obesidade é considerada uma doença crônica e multifatorial, onde eventos como a inflamação de baixa intensidade e as modificações oxidativas estão presentes. A elevada prevalência de obesidade tem impacto direto no desenvolvimento precoce de diabetes mellitus, hipertensão e outros fatores de risco cardiovasculares. Esse perfil tem motivado a identificação de biomarcadores precoces, sendo o monitoramento da lipoproteína de baixa densidade oxidada (LDLox) e lipoproteína de baixa densidade eletronegativa [LDL(-)] potenciais candidatos. Diante do exposto, o objetivo deste estudo foi realizar a análise comparativa e de correlação entre o conteúdo de LDLox e [LDL(-)] em adolescentes. Foram selecionados 137 adolescentes de ambos sexos, com faixa etária de 10 a 19 anos e regularmente em matriculados em escolas públicas da cidade de São Paulo. O peso, altura e circunferência da cintura (CC) foram avaliados. Após jejum (12h-15h) foi coletada uma amostra de sangue e, a partir do plasma, foram realizadas as seguintes análises: glicose, insulina, perfil lipídico, apolipoproteína (AI e B), ácidos graxos não esterificados, tamanho de HDL, atividade da CETP e LDL(-) e LDLox. Os resultados encontrados foram analisados por meio do programa SPSS 15.0, considerando valor de significância de p< 0,05. Os 137 adolescentes foram distribuídos em dois grupos: 71 no Eutrófico (51,82%) e 66 no Obeso (48,18%), segundo a classificação do IMC. 48 (35,04%) dos adolescentes eram do sexo masculino e 89 (64,96%) do sexo feminino, com idade média de 14,2 (2,3) anos. Em relação à CC, observou-se que essa confirmou a classificação feita pelo IMC. Observou-se também uma maior prevalência de hipertensão (65% p = 0,011) e obesidade (64,7% p=0,041) nos antecedentes familiares do grupo Obeso quando comparado ao grupo Eutrófico. Os adolescentes obesos apresentaram maiores valores de triglicerídeos, HDL, APO B, CETP, insulina e LDL(-) e LDLox, quando comparados aos eutróficos. Perfil inverso foi observado para Apo AI. O conteúdo de LDLox e LD(-) variou significativamente em função do IMC. Entretanto, essas partículas de LDLs não se correlacionaram entre si, embora tenham apresentado associação com outros parâmetros cardiometabólicos. Os resultados obtidos confirmam o impacto negativo da obesidade sobre os parâmetros cardiometabólicos de adolescentes e, apesar do conteúdo de LDLox e LDL(-) ter aumentado em função do IMC, essas partículas parecem ser estruturalmente distintas. Essa possibilidade foi reforçada pelas diferentes associações dessas partículas com outros marcadores bioquímicos. / Obesity is considered a chronic and multifactorial disease, where events such as low intensity inflammation and oxidative modifications are present. The high prevalence of obesity has a direct impact on the early development of diabetes mellitus, hypertension and other cardiovascular risk factors. This profile has motivated the identification of early biomarkers, and monitoring the oxidized low density lipoprotein (oxLDL) and electronegative low-density lipoprotein [LDL (-)] are potential markers. The aim of this study was to conduct a comparative analysis and correlation between the content of oxLDL and [LDL (-)] in adolescents. We selected 137 adolescents of both sexes, aged 10-19 years, enrolled in public schools in the city of São Paulo. The weight, height, waist circumference (WC) were assessed. After fasting (12-15h) samples of blood were collected and from plasma were performed the following analyzes: glucose, insulin, lipid profile, apolipoprotein (AI and B), NEFA, HDL size, CETP activity and LDL (-) and oxLDL. The results were analyzed by using SPSS 15.0 considering significant value of p <0.05. The 137 adolescents were divided into two groups: 71 Normal Weight (51.82%) and 66 Obese (48.18%), according to BMI classification. 48 (35.04%) of the adolescents were male and 89 (64.96%) females with a mean age of 14.2 (2.3) years. Regarding the CC observed, it confirmed the classification made by BMI. There was a higher prevalence of hypertension (65% p = 0.011) and obesity (64.7% p = 0.041) in the family history group Obese when compared to normal weight. Obese adolescents had higher triglyceride, HDL, APO B, CETP, insulin and LDL (-) and oxLDL, compared to normal weight. Reverse profile was observed for Apo AI. The content of oxLDL and LDL (-) varied significantly according to BMI. However, these LDL particles were not correlated with each other, although they showed cardiometabolic combination with other parameters. The results confirm the negative impact of obesity on cardiometabolic parameters of teenagers and although the content of oxLDL and LDL (-) increased as a function of BMI, these particles appear to be structurally distinct. This possibility was reinforced by different associations of these particles with other biochemical markers.

The role of cell death in atherosclerosis remains ill-defined, however, a growing body of evidence suggests that cell death stimulates atherogenesis through the induction of inflammation and enlargement of the necrotic core. Although there is solid evidence to suggest that lipid oxidation and toxicity are linked, indications that protein oxidation may play an important role in cytotoxicity are numerous. The abundance of dead cells in atherosclerotic plaques and their co-localization with HOCl-modified proteins provides an opening for the suggestion that the products of protein oxidation may be at the heart of oxLDL-induced cell death. Examination of the modification of LDL and albumin by HOCl, and the cytotoxicity of these oxidised molecules were the focus of this study, along with the elucidation of their cell death mechanisms toward U937 cells. Measurement of lipid peroxidation markers, TBARS and 7-ketocholesterol, showed no significant increase in HOCl-oxLDL compared to native levels although all α-tocopherol had been lost. In contrast there was a large loss of tyrosine, of which a small percentage went to dityrosine, indicating that the protein moiety of LDL was the main target of HOCl attack. Albumin became fragmented and smeared on SDS-PAGE gels with increasing HOCl/BSA molar ratios. In addition there was significant reduction in tyrosine levels and a small increase in dityrosine. Both HOCl-oxLDL and oxidised albumin (oxALB) caused concentration-dependent cell viability loss in U937 cells following a significant drop in intracellular GSH concentration, coinciding with a peak in oxidative stress. Removal of chloramines with methionine significantly reduced the toxicity of oxALB, but at higher concentrations this effect was reduced. This was in contrast to HOCl-oxLDL where the removal of chloramines had no effect on its toxicity. Morphological observations of cell swelling, cell membrane integrity loss and rupture, along with flow cytometry results indicate that U937 cells underwent necrosis, but only after intracellular GSH was lost. Intracellular GSH and cell viability loss were prevented by 200 μM extracellular 7,8-dihydroneopterin (78NP), indicating that 78NP scavenging of ROS generated in response to the oxidised proteins was sufficient to prevent cell death. This study demonstrates the cytotoxicity of HOCl-damaged LDL and albumin is likely due to a common oxidative product or structural motif which may be active within atherosclerotic plaques.

Proefschrift Universiteit van Amsterdam. / Met lit. opg. - Met samenvatting in het Nederlands.

Proprotein Convertase Subtilisin / Kexin Type-9 (PCSK9) has emerged as a major regulator of plasma cholesterol levels. PCSK9 is secreted mainly from the liver and circulates as a plasma protein. PCSK9 binds cell surface low-density lipoprotein (LDL) receptors and mediates their degradation upon endocytosis in the liver. This decreases the liver’s ability to clear LDL-cholesterol from the blood. PCSK9 is also capable of binding LDL particles themselves; this interaction inhibits the ability of PCSK9 to bind and mediate LDLR degradation in cultured hepatic cells, but its effect on PCSK9 function in vivo remains unknown. A disordered N-terminal region of the PCSK9 prodomain is necessary for binding to isolated LDL particles in vitro. This N-terminal region is also autoinhibitory to PCSK9-LDL receptor binding. We hypothesized that the N-terminal of the PCSK9 prodomain plays a role in an allosteric mechanism that regulates PCSK9 function. Through mutagenesis studies, we found that both a conserved stretch of acidic residues and an adjacent conserved stretch of hydrophobic residues are crucial for the PCSK9-LDL interaction; the hydrophobicity of the residue at position 38 (Tyr) within the conserved acidic stretch was also found to be important for this. Helical wheel modeling of the prodomain N-terminal sequence revealed the potential for a lipid-ordered amphipathic helix to form, which may aid PCSK9 docking onto LDL. Replacing residues A44 and L41 with helix-disrupting proline residues abolished LDL binding. Co-pelleting ultracentrifugation assays also show that wild-type PCSK9 is capable of associating with liposomes, while the A44P mutation disrupts this lipid association. The A44P-PCSK9 mutation, showing an 80-90% decrease in LDL association but with LDL receptor binding and degrading functions intact, may serve as an important tool in future studies investigating the PCSK9-LDL interaction in vivo. Elucidation of the mechanism by which LDL-binding naturally inhibits PCSK9 activity may also help to develop new anti-PCSK9 therapeutics in the future.